Abstract

Treatment with lamivudine of patients infected with hepatitis B virus (HBV) results in a high rate of drug resistance, which is primarily associated with the rtM204I/V substitution in the HBV reverse transcriptase domain. Here we show that the rtM204I/V substitution, although essential, is insufficient for establishing resistance against lamivudine. The analysis of 639 HBV whole-genome sequences obtained from 11 patients shows that rtM204I/V is independently acquired by more than one intra-host HBV variant, indicating the convergent nature of lamivudine resistance. The differential capacity of HBV variants to develop drug resistance suggests that fitness effects of drug-resistance mutations depend on the genetic structure of the HBV genome. An analysis of Bayesian networks that connect rtM204I/V to many sites of HBV proteins confirms that lamivudine resistance is a complex trait encoded by the entire HBV genome rather than by a single mutation. These findings have implications for public health and offer a more general framework for understanding drug resistance.

The node size is proportional to frequency of the corresponding variant in the viral population. Colours correspond to the codon states at position rt204: rtM204I coded by ATT or ATC is shown in red or grey, respectively; rtM204V is shown in blue (codon GTG); the wild type is shown in cyan (codon ATG). The distance between the two genotype clusters of Patient 1 was plotted 20 times shorter to allow the visualization of minor variants.

Maximum likelihood trees of intra-host HBV variants sampled before and after lamivudine therapy.

HBV variants were sampled from Patients 6–11 before (blue) and after (red) treatment with lamivudine. The node size is proportional to frequency of the corresponding variant in the viral population. For Patients 8 and 9, only pre-treatment subpopulations that were genetically closest to the resistant variants are shown.

Scatterplot of distances among intra-host HBV variants of six patients.

Genetic distances are shown for Patients 6–11. Each pre-lamivudine sequence was plotted according to its value in two dimensions: the average distance to all other pre-treatment variants (x axis) and the average distance to all other post-treatment variants (y axis).